Field of the Invention
The present invention relates to the detecting and analyzing instrument, especially to both ultraviolet-visible and double monochromatic fluorescence dual detector for high performance liquid chromatography.
Description of Related Arts
The liquid chromatograph is an instrument for detecting and analyzing organic compound samples quantitatively and qualitatively, and is widely used in petrochemical, medical care, environment protection, food, traditional Chinese/western medicine, beverage, biochemics, and health products etc. to detect and analyze the component of the sample. At present, liquid chromatography mainly comprises high performance liquid chromatography, ion chromatography, capillary chromatography, gel permeation chromatography and the like, wherein high performance liquid chromatography (HPLC) is most widely used.
Liquid chromatography mainly includes a high pressure infusion pump, a high pressure sampling system, a chromatographic column, various detectors and a data processing system connected with the computer. Before the analyzing and detecting process, firstly various mobile phases should be mixed, stirred and degassed, then the sample is prepared into a sample solution which enters into the high pressure sampling system and is delivered into the chromatographic column through the high pressure infusion pump. Since each component of the sample solution has different partition coefficient in two phases, after many times of absorption-desorption partition process, the sample can be separated, as per different time passing the chromatographic column, into chromatographic peak of individual component for the convenience of qualitative analysis, which flowed out in turn from the chromatography column and entered directly into the detector. In the detector, the concentration of each component of the sample is converted to different voltage signals by the detector. After amplifying, zero setting and other processing arts, the output voltage signals are connected with a computer through the data processing system, and the analysis results of the individual component of the sample are saved, displayed and printed in the form of chromatogram and data. From the above content, the stability and detectability of the detector is the key of the whole chromatography instrument. The technical performance of the detector directly decides the technical performance of the whole chromatography instrument, and is an important prerequisite of the accurately qualitative and quantitative analysis of the sample.
In the prior art, ultraviolet visible detector is the earliest as well as the most widely used detector, but it still has heavy technical problems so far: the existing ultraviolet visible detectors cannot overcome or solve the objective existing worldwide technical difficulties: 1. The photoelectric signals generated by the three major components in the optical member of the instrument—lighting lamp, monochromatic device-raster, and receiving photosensitive diode are quite different from each other due to the variation with different wavelengths, and the instrument operates in accordance with Beer-Lambert Law: absorption unit AU=LOG photoelectric signal of incident light/photoelectric signal of transmission light=absorption coefficient×sample concentration (after logarithm), among which the denominator on the right of the equal sign is unknown and the numerator is variable, so none of the existing products can calculate the AU value for each wavelength in the whole wavelength range? As a result, absorption unit AU value of only one optical wavelength can be inspected and calculated, while that of other hundreds of wavelengths cannot be calculated, calibrated, inspected, or checked correctly, but main technical targets in noise and drift indicating the stability of the instrument are calculated and calibrated by absorption unit AU value; as more than 99% of the objective existing wavelengths cannot be calibrated or checked correctly and no technical methods are adopted, technical targets of one wavelength is used to act as different technical targets of other hundreds of wavelengths, and the personnel of inspection agencies are not able to inspect or check so many wavelengths; 2. As there is no special art other than general amplifying processing for weak photoelectric signals, and the simultaneous amplifying of signal together with noise and drift cannot solve the long existing worldwide technical difficulty of the mutual exclusivity and contradiction between sensitivity and stability (i.e. failing to increase the signal to noise ratio), the existing products in the existing production or use have low sensitivity and stability. Therefore, the inspection of samples that should be inspected are often neglected, and it is inevitable that the products are actually not as good as they are described in the publicity documents, and some instruments with wavelength range (190˜210 NM) even does not conform to the detection standards and cannot be used, and are actually the “defective products” that cannot be used in the whole wavelengths. More notably, scientific and technical personnel have found that a great number of “DAD diode array ultraviolet detectors” to be used for high performance liquid chromatography, which are produced in some countries and exported to universities and research detection institutions in different countries across the world, actually go against and misuse the theory of “Beer-Lambert Law” studied in physics of middle school: first, monochromatic device without the front of sample detection cell (which is indispensable in practice as well as classical instrument) are introduced in the past trade fairs, and this can be proved by actual products; wavelengths of the front of sample detection cell can only be replaced with those of the back of sample detection cells after spectroscopical process. Actually, this kind of misuse and faking can prove to be the only choice by theories and practice, because the classical and correct set should be the monochromatic component of the front of sample detection cell, and only one photosensitive diode, instead of array of hundreds of photosensitive diodes, is needed to stay close to the back of the detection cell, which shows that components of the array have no practical meaning; secondly, the hybrid light of various wavelengths in front of the sample detection cell enters to the sample detection cell, and actually turns technical targets of optical instruments such as the accuracy, precision, and repeatability of wavelength into dummy false targets and those instruments won't become real optical instruments; thirdly, a lot of non-analytical wavelength's light also have different absorption by the sample and thus generate false photoelectric signals, then such signals will act as analysis wavelength's photoelectric signal and create a hoax of high sensitivity (it can be proved by two absorption peak signals of different wavelengths both sensitive to the same naphthalene sample in Embodiment 1 in Specification); fourth, adding a lot of absorption coefficients of non-analysis wavelengths will lead to relatively large errors in calibration curve compared with that of the classical and standard products, causing the mistake that the result of quantitative analysis is different from that of the classical products; fifth, more chromatographic peaks of other wavelengths to unknown impurities in sample will be generated, and it will be mistaken for high detectting ability.
Fluorescence spectroscopical detector can be classified into: spectroscopical component with low-grade of one monochromator and high-grade of two monochromators. As the high-grade double monochromator spectroscopical component is far difficult than the single monochromator component of ultraviolet detector, the photocurrent signal generated by the secondary emission wavelength will be extremely weak (lower than that of the ultraviolet by 4˜5 orders of magnitude). Therefore, smaller signal-to-noise ratio and extremely bad stability will be generated, causing great difficulty in development, and it is hard to meet necessary standards in technical performance. This explains that no products used in the liquid chromatographic high-grade fluorescence have been successfully developed in China, and fluorescence detectors with high performance in liquid chromatograph containing double monochromator are also rarely seen in other countries. Most of them are low-grade fluorescence detector with only one monochromator. In addition, for such detector has no special art in the elimination of noise and drift to increase signal-to-noise ratio, there is no doubt that the technical performance is worse than that of ultraviolet. The excitation wavelength or the emission wavelength cannot be correctly decided even if sample of high concentration are needed for the detection of chromatographic peaks to appear. These two wavelengths should be known, but in fact now one of them is unknown, which brings great difficulties to researchers conducting qualitative analysis of many unknown samples.